Phosphorylation has come to be recognized as a global regulator of cellular activity, and abnormal phosphorylation is implicated in a host of human diseases, particularly cancers. Phosphorylation of a protein involves the enzymatically mediated addition of a phosphate group (—OPO3−2) to its amino acid side chains of one or more serine, threonine or tyrosine residues.
Phosphorylation and the reverse reaction, dephosphorylation, occur via the actions of two key enzymes. Protein kinases phosphorylate proteins by transferring a phosphate group from a nucleotide triphosphate such as adenosine triphosphate (ATP) to their target protein. This process is balanced by the action of protein phosphatases, which can subsequently remove the phosphate group. The amount of phosphate that is associated with a protein at a particular time is therefore determined by the relative activities of the associated kinase and phosphatase.
If the protein is an enzyme, phosphorylation and dephosphorylation can impact its enzymatic activity, essentially acting like a switch, turning it on and off in a regulated manner. Phosphorylation can regulate protein-protein interactions by facilitation of binding to a partner protein.
Protein phosphorylation also has a vital role in intracellular signal transduction. Many of the proteins that make up a signaling pathway are kinases, from the tyrosine kinase receptors at the cell surface to downstream effector proteins, many of which are serine/threonine kinases.
Not all targets of phosphorylation are proteins. Other kinds of molecules can also be phosphorylated. In particular, the phosphorylation of phosphoinositide lipids, such as phosphatidylinositol-4,5-bisphosphate (PIP2), at various positions on their inositol ring, also plays a key role in signal transduction.
Thus, ligand binding at the cell surface can establish a phosphorylation cascade, with the phosphorylation and activation of one protein stimulating the phosphorylation of another, subsequently amplifying a signal and transmitting that signal through the cell. The signal continues to propagate until it is switched off by the action of a phosphatase.
Recent research activity has successfully developed drugs that interact with kinase and phosphatase activities. Illustrative of approved drugs and their target proteins are Herceptin® that blocks the function of the HER2-receptor (used in breast cancer treatment), Tarceva® that inhibits the epidermal growth factor receptor (EGFR) that is a tyrosine kinase (used in non-small cell lung cancer treatment), Afinitor® and Torisel® target the mammalian target of rapamycin (mTOR) (used in renal carcinoma treatment). Drugs in the pipeline to approval include Keryx™ that is an inhibitor of Akt [also known as protein kinase B (PKB)], selumetinib that targets MEK (also known as MAPKK), and alvocidib that targets cyclin-dependent kinases.
Human phosphatase and tensin homolog deleted on chromosome ten (PTEN; EC 3.1.3.16; UniProt Accession P60484) is a protein enzyme that, in humans, is encoded by the PTEN gene. [Steck et al., Nat. Genet. 15(4):356-362 (1997).] The corresponding PTEN protein is found in almost all tissues in the body in both the cytoplasm and the nucleus and acts as a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase.
As a lipid phosphatase, PTEN dephosphorylates phosphatidylinositol (3,4,5)triphosphate [PtdIns (3,4,5)P3 or PIP3] to form the corresponding diphosphate, phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2 or PIP2). PTEN is a lipid-second messenger and a regulator of the PI3K/Akt pathway [Wu al., Oncogene 22:3113-3122 (2003)] thereby playing a central role in controlling many important cellular activities regulated by this pathway, including cell division, cell growth, cell survival, and DNA damage.
The dephosphorylation of PIP3 is important because it leads to decreased phosphorylated-Akt (P-Akt) levels resulting in inhibition of the AKt signaling pathway; i.e., inhibition of Akt phosphorylation. PTEN's protein phosphatase activity (PPA) is also known to regulate cyclin D1 protein levels and those activities appear to be regulated by nuclear PTEN [Chung et al., Cancer Res. 65:8096-8100 (2005)].
The PTEN gene acts as a tumor suppressor gene through the action of its phosphatase protein product that dephosphorylates both protein and lipid substrates. This phosphatase is involved in the regulation of the cell cycle, preventing cells from growing and dividing too rapidly. [Chu et al., Med. Sci. Monit. 10 (10): RA235-241 (2004).] It is one of the targets of an oncomiR, miRN21, a microRNA.
The PTEN gene is mutated at high frequency in a large number of cancers. Thus, loss of functionally active PTEN, as demonstrated by genetic inactivation in human cancer or mouse knockout (KO) models, causes constitutive activation of Akt in cells, resulting in dysregulated cell proliferation, growth, and survival, which are hallmarks of tumorigenesis.
The structure of PTEN [solved by X-ray crystallography; Lee et al., Cell 99(3):323-334 (1999)]reveals that it consists of a phosphatase domain, and a C2, tensin-like, domain, providing a structural similarity to the dual specificity protein tyrosine phosphatases. The phosphatase domain contains the active site, which carries out the enzymatic function of the protein, whereas the C2 domain binds the phospholipid membrane. Thus, PTEN binds the membrane through its C2 domain, bringing the active site to the membrane-bound PIP3 to de-phosphorylate it.
Unlike most of the protein tyrosine phosphatases, this protein preferentially dephosphorylates phosphoinositide substrates. PTEN protein negatively regulates phosphatidylinositol-3,4,5-trisphosphate (PIP3) in cells and functions as a tumor suppressor by negatively regulating the Akt/PKB signaling pathway. PTEN negatively regulates phosphorylation of phosphatidylinositol 4,5-bisphosphate by phosphoinositide 3-kinase (PI3K), a main regulator of cell growth, metabolism and survival. PI3K-mediated PIP(3) production leads to the activation of the canonical Akt-mTORC1 pathway.
Akt is activated by PDK1-mediated phosphorylation at Thr308 and by phosphorylation at Ser473 via mTOR complex 2 [mTORC2; composed of mTOR, DEP domain-containing mTOR-interacting protein (DEPTOR), mammalian lethal with SEC13 protein 8 (mLST8), stress-activated MAP kinase-interacting protein 1 (mSIN1), Pro-rich protein 5 (PRR5; also known as PROTOR) and rapamycin insensitive companion of mTOR (RICTOR)]. Active Akt drives cell survival, proliferation and cellular metabolism through inhibiting phosphorylation of downstream proteins, including glycogen synthase kinase 3 (GSK3), forkhead box O (FOXO), peroxisome proliferator-activated receptor-γ (PPARγ) co-activator 1α (PGC1) and p27, and through activatory phosphorylation of ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5), sterol-responsive element-binding protein 1C (SREBP1C), AS160 and S phase kinase-associated protein 2 (SKP2). Akt can also activate mTORC1 [composed of mTOR, DEPTOR, mLST8, 40 kDa Pro-rich Akt1 substrate 1 (PRAS40) and regulatory associated protein of mTOR (RAPTOR)] by mediating the inhibitory phosphorylation of its negative regulators tuberous sclerosis protein 2 (TSC2) and PRAS40.
When the PTEN enzyme is functioning properly, it acts as part of a chemical pathway that signals cells to stop dividing and can cause cells to undergo programmed cell death (apoptosis) when necessary. These functions prevent uncontrolled cell growth that can lead to the formation of tumors. There is also evidence that the protein made by the PTEN gene may play a role in cell movement (migration) and adhesion of cells to surrounding tissues.
PTEN is one of the most commonly lost tumor suppressors in human cancers. During tumor development, mutations and deletions of PTEN occur that inactivate its enzymatic activity leading to increased cell proliferation and reduced cell death.
Frequent genetic inactivation of PTEN occurs in glioblastoma, endometrial cancer, melanoma, small cell lung cancer and prostate cancer; and reduced expression is found in many other tumor types such as lung and breast cancer. “Up to 70 percent of men with prostate cancer have lost one copy of the PTEN gene at the time of diagnosis”. [Chen et al., Nature 436 (7051):725-730 (2005).]
PTEN mutations have also been found in non-cancerous syndromes. For example, researchers have found more than 70 mutations in the PTEN gene in people with Cowden syndrome. These mutations can be changes in a small number of base pairs or, in some cases, deletions of a large number of base pairs. Most of these mutations cause the PTEN gene to make a protein that does not function properly or does not work at all. The defective protein is unable to stop cell division or signal abnormal cells to die, which can lead to tumor growth, particularly in the breast, thyroid, or uterus.
Mutations in the PTEN gene cause several other disorders that, like Cowden syndrome, are characterized by the development of noncancerous tumors called hamartomas. These disorders include Bannayan-Riley-Ruvalcaba syndrome, and Proteus-like syndrome. Together, the disorders caused by PTEN mutations are called PTEN hamartoma tumor syndromes, or PHTS. Mutations responsible for these syndromes cause the resulting protein to be nonfunctional or absent. The defective protein allows the cell to divide in an uncontrolled way and prevents damaged cells from dying, which can lead to the growth of tumors.
Defects of the PTEN gene have been cited to be a potential cause of Autism Spectrum Disorders. [Napoli et al., PLoS One 7(8):e42504 (2012).] When defective, PTEN protein interacts with the protein of a second gene known as p53 to dampen energy production in neurons. This severe stress leads to a spike in harmful mitochondrial DNA changes and abnormal levels of energy production in the cerebellum and hippocampus—brain regions critical for social behavior and cognition. When PTEN protein was insufficient, its interaction with p53 has been shown to trigger deficiencies and defects in other proteins that also have been found in patients with learning disabilities including autism. [Napoli et al., PLoS One 7(8):e42504 (2012).]
The RAS proteins are members of a large superfamily of low molecular-weight GTP-binding proteins, which can be divided into several families according to the degree of sequence conservation. Different families are important for different cellular processes—the RAS family controls cell growth and the RHO family controls the actin cytoskeleton. Three members of the RAS family—HRAS, KRAS and NRAS—are found to be activated by mutation in human tumors [Lowy et al., Annu. Rev. Biochem. 62:851-891 (1993)]. These three members are very closely related, having 85 percent amino acid residue sequence identity, and although they function in very similar ways, some indications of subtle differences between them have recently come to light.
The HRAS, KRAS and NRAS proteins are widely expressed, with KRAS being expressed in almost all cell types. Knockout studies have shown that Hras and Nras, either alone or in combination, are not required for normal development in the mouse, whereas Kras is essential [Johnson et al., Genes Dev. 11:2468-2481 (1997)]. The normal function of RAS proteins requires them to be post-translationally modified.
RAS proteins have essential roles in controlling the activity of several crucial signaling pathways that regulate normal cellular proliferation. Ras mediates downstream signaling by interacting with effector proteins, including Raf and PI3 kinase.
Raf1 is a serine/threonine protein kinase that is part of the MAP kinase signaling pathway that leads to the activation of ERK and p38, which influences proliferation and survival. Signaling by PI3 kinase activates AKT and mTOR, which are central for cell growth and survival. Ras is also integral for cellular differentiation and development, including immune cell development and function.
Human tumors very frequently express RAS proteins that have been activated by point mutation—about 20 percent of all tumors have undergone an activating mutation in one of the RAS genes [Bos, Cancer Res. 49:4682-4689 (1989)]. In these tumors, the activated RAS protein contributes significantly to several aspects of the malignant phenotype, including the deregulation of tumor-cell growth, programmed cell death (apoptosis) and invasiveness, and the ability to induce new blood-vessel formation [Shields et al., Trends Cell Biol. 10:147-154 (2000)].
The activation state of RAS proteins depends on whether they are bound to GTP (in which case, they are active and are able to engage downstream target enzyme) or GDP (in which case, they are inactive and fail to interact with these effectors). In normal, non-cancerous cells, the activity of RAS proteins is controlled by the ratio of bound GTP to GDP [Campbell et al., Oncogene 17:1395-1413 (1998)]. In vitro, purified RAS possesses a low level of intrinsic GTPase activity; i.e., bound GTP is slowly converted to GDP. It also has a slow rate of nucleotide exchange with the surrounding medium; i.e., bound GDP is gradually replaced by GTP. However, these processes are catalyzed within the cell by guanine nucleotide exchange factors (GEFs) and the nucleotide hydrolysis by GTPase activating proteins (GAPs). Both of these activities involve large, considerably divergent families of proteins. It is the balance between these proteins that determines the activation state of RAS and its downstream target pathways. [Downward, Nature Reviews Cancer 3:11-22 (2003).]
In addition to PTEN and Kras, a growing body of research is revealing a complex role of filamin A (FLNA) in cancer. [Yue et al., Cell & Bioscience 3:7 (2013).] Best known for cross-linking cytoplasmic actin into dynamic scaffolds to control cell motility, filamins are large cytoplasmic proteins increasingly found to regulate cell signaling by interacting with over 30 different receptors and signaling molecules [Feng et al., Nat Cell Biol 6:1034-1038 (2004); Stossel et al., Nature Rev. Mol. Cell Biol. 2:138-145 (2001)], including the mu opioid receptor (MOR) [Onoprishvili et al., Mol Pharmacol 64:1092-1100 (2003)]. Filamins are dimerized through the last carboxy-terminal repeat near the transmembrane regions, allowing an intracellular V-shaped structure that is critical for function. There are three mammalian isoforms: filamin A (FLNA), B and C. As a key regulator of the cytoskeleton network, FLNA interacts with many proteins involved in cancer metastasis, [Yue et al., Cell & Bioscience 3:7 (2013)] and its knockdown or inhibition has been shown to reduce cancer metastasis potential. [Xi et al., Int J Biol Sci 9:67-77 (2013).]
Human FLNA is given the identifier P21333 in the UniProtKB/Swiss-Prot data base, and contains a sequence of 2647 amino acid residues. This protein is also sometimes referred to in the art as actin-binding protein (ABP-280). [Gorlin et al., J Cell Biol 111:1089-1105 (1990).] Nakamura et al., Cell Adh Migr. 5(2):160-169 (2011) discuss the history of research concerning FLNA and notes that the protein serves as a scaffold for over 90 binding partners including channels, receptors, intracellular signaling molecules and transcription factors.
FLNA also has been implicated in tumor progression. FLNA knockout mice show reduced oncogenic properties of K-RAS, including the downstream activation of ERK and Akt. [Nallapalli et al., Mol Cancer 11:50 (2012).] Many different cancers show high levels of FLNA expression in contrast to low FLNA levels in corresponding normal tissue, including colorectal and pancreatic cancer, [Uhlen et al., Mol Cell Proteomics 4:1920-1932 (2005)] and glioblastoma [Sun et al., Cancer Cell 9:287-300 (2006)]. Inhibition of FLNA also sensitizes cancer cells to both cisplatin and radiation [Sun et al., Cancer Cell 9:287-300 (2006)], and FLNA deficiency in cancer cells similarly sensitizes them to chemotherapeutic agents [Yue et al., DNA Repair (Amst) 11:192-200 (2012)] and radiation [Yue et al., Cancer Res 69:7978-7985 (2009); Yuan et al., J Biol Chem 276:48318-48324 (2001)]. On the other hand, Jiang et al., Int. J. Biol. Sci. 9:67-77 (2013) report that inhibition of filamin A expression leads to reduced metastasis in nude mice implanted with melanoma and breast cancer cells.
Additionally, FLNA is phosphorylated at a number of positions in its protein sequence in both normal and in cancer cells. For example, FLNA is phosphorylated at position 2152 by PAK1 as is required for PAK1-mediated actin cytoskeleton reorganization and for PAK1-mediated membrane ruffling. [Vadlamudi et al., Nat. Cell Biol. 4:681-690 (2002); Woo et al., Mol Cell Biol. 24(7):3025-3035 (2004).] Cyclin B1/Cdk1 phosphorylates serine 1436 in vitro in FLNA-dependent actin remodeling. [Cukier et al., FEBS Letters 581(8):1661-1672 (2007).]
The UniProtKB/Swiss-Prot data base entry for human FLNA (P21333) lists published reports of the following amino acid residue positions as being phosphorylated under different circumstances: 11, 1081, 1084, 1089, 1286, 1338, 1459, 1533, 1630, 1734, 2053, 2152, 2158, 2284, 2327, 2336, 2414, and 2510. Further, polyclonal and monoclonal antibodies are commercially available from one or more of Abgent, Inc. (San Diego, Calif.), Abcam, Inc. (Beverly, Mass.), Bioss, Inc. (Woburn, Mass.), and GeneTex, Inc. (Irvine, Calif.) that immunoreact with FLNA that is phosphorylated (phospho-FLNA) at serine-1083, tyrosine-1046, serine-1458, serine-2152, and serine-2522.
Inflammation is implicated in tumorgenesis, metastasis and angiogenesis [Coussens et al., Nature 420:860-867 (2002)], and FLNA can regulate inflammation via its recruitment to toll-like receptor 4 (TLR-4) that is required for signaling and subsequent release of inflammatory cytokines IL-6, IL-1β and TNFα [Wang et al., J Neurosci 32:9773-9784 (2012)]. Activation of TLRs on cancer cells promotes chronic inflammation which stimulates cancer cell proliferation, migration, tumor angiogenesis, and creates a tumor microenvironment that impairs the antitumor function of the immune system, allowing tumors to develop and survive [McCall et al., “Toll-Like Receptors as Novel Therapeutic Targets for the Treatment of Pancreatic Cancer” (Chapter 20), Pancreatic Cancer—Molecular Mechanism and Targets, Srivastava Ed., InTech, Rieka, Croatia, 361-398 (2012)].
In particular, the inflammatory cytokines TNFα, IL-6 and IL-1β have been implicated in tumor growth and angiogenesis [Leibovich et al., Nature 329:630-632 (1987); Waters et al., J Pathol 230:241-248 (2013); Wei et al., Oncogene 22:1517-1527 (2003); Voronov et al., Proc Natl Acad Sci, USA 100:2645-2650 (2003); Carmi et al., J Immunol 190:3500-3509 (2013); Naldini and Carraro In: Current Drug Targets—Inflammation & Allergy, 4:3-8 (2005); Shchors et al., Genes Dev 20:2527-2538 (2006)]. Because the release of these inflammatory cytokines is controlled predominantly by TLR4 and TLR2, disrupting the required association of FLNA with these receptors can suppress angiogenesis and tumor growth.
The role of FLNA and phospho-FLNA in cellular biology of both normal, disease-free states and in cancerous states is seen to be quite complex and not yet definitively worked out. The present invention and its underlying biochemical bases disclosed hereinafter help to further delineate some of the biochemical pathways involved in the cancerous condition of some cells and assist in treating that condition to inhibit the progression of the cancerous state.